CN109266710A - Production method of pig foot-and-mouth disease O-type genetic engineering composite epitope protein vaccine - Google Patents
Production method of pig foot-and-mouth disease O-type genetic engineering composite epitope protein vaccine Download PDFInfo
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Abstract
The invention relates to an industrialized production method of a pig foot-and-mouth disease O-type genetic engineering composite epitope protein vaccine, which comprises the following steps in sequence: (1) fermenting and inducing the engineering bacteria to express; (2) collecting and crushing thalli; (3) washing and denaturing dissolving of inclusion body; (4) purifying the protein; (5) renaturation of the purified protein; (6) and (4) emulsifying the vaccine. The invention has the beneficial effects that: the product quality aspect is as follows: the vaccine is sterile, the purity of target protein is more than 95%, the content of effective antigen is more than 1.0mg/mL, and the indirect hemagglutination inhibition titer is 1: 211Above, endotoxin is below 31.25EU/mL, production cost and efficiency are as follows: less personnel, less reagent consumption and shorter production period.
Description
Technical field
The invention belongs to immunological technique fields, and in particular to a kind of Schweineseuche O-shaped genetic engineering multi-epitope albumen
Production method.
Background technique
Aftosa (Foot-and-Mouth Disease, FMD) is by foot and mouth disease virus (Foot-and-Mouth
Disease Virus, FMDV) caused by artiodactyl deadly infectious disease, be World Organization for Animal Health (OIE) regulation must report
Deadly infectious disease, China is arranged in first of a kind of infectious disease.Foot and mouth disease virus shares seven serotypes, respectively O, A,
C, Asia I, SAT1, SAT2, SAT3 type, it has been found that in China, prevalence has I type of O, A and Asia, and O-shaped aftosa is to endanger at present
Evil serotype the most serious, epidemic situation is in successive years, causes serious economic loss.Currently, the O-shaped mouth hoof existing for China
Three epidemic disease prevalence Du You Burma 98 (Mya98), Pan Asia (PanAsia) and sinotype (Cathay) pedigrees, wherein 98 system of Burma flows
Row is the most extensive.The generation of the disease seriously hinders the development of social economy with popular, restricts healthy aquaculture, is that solution is badly in need of in China
One of major issue certainly.
China takes vaccine immunity with slaughtering the aggregate measures prevention and control aftosa combined, country freely to all domestic animals into
Row compulsory immunization.Predominantly inactivated vaccine used at present.But because foot and mouth disease virus immunogenicity itself is weak, the immune of vaccine is held
It renews shorter, needs repeatedly immune, immune animal can generate 3ABC antibody, be unfavorable for infection and immune identification.In addition, inactivation
The generation of vaccine needs high level safeguard procedures to eliminate the security risks such as viral escape.Due to these factors, promote people
Develop the Vaccine for hoof-and-mouth disease of highly effective and safe.
Foot-and-mouth disease VP1G-H ring and its C- terminal sequence are most important linear neutralizing epitopes, pass through expression
Or with synthesize this peptide fragment immune cattle cannot obtain complete immunoprotection (Taboga et al., 1997； Wang et al.,
2002；Rodriguez et al.,2003；Zhang et al.,2015).But with similar synthetic peptide vaccine immune swine body
Be capable of providing to the preferable immune protective efficiency of homologous virus (Taboga et al., Wang et al., 2002；Shao et
al.,2011；Zhang et al.,2015).
This researching and designing multiple epitopes concatenated immunogen genes, the sinotype for covering China's history and currently occurring
(2 subbreed), Pan Asia system and the O-shaped main neutrality epitope area of foot and mouth disease poison of 98 3 pedigrees of Burma, in addition Universal T-cell
Mouse is immunized in prokaryotic cell, picks out immunogenicity and the best albumen of cross reactivity for epitope after expression, purification renaturation
As seedling antigen, using the CpG of the mature differentiation of inducing dendritic shape cell (DC) as immunopotentiator, the two compatibility is simultaneously passed through
Vaccine is made in the emulsification of ISA201VG oil adjuvant, has developed Schweineseuche O-shaped multi-epitope protein vaccine.It is inactivated with existing aftosa
Vaccine is compared, and poison living is not employed in the production of Schweineseuche O-shaped multi-epitope protein vaccine, that is, does not need inactivation technology, without mouth
Fever aphthous totivirus nucleic acid, it is very safe；This vaccine does not contain any foot and mouth disease virus non-structural protein ingredient yet, i.e., repeatedly immune
Non-structural protein antibody will not be all generated, avoids the interference to aftosa natural infection and vaccine immunity antidiastole, and raw
Produce relative inexpensiveness.CpG immunopotentiator, (the Cao et compared with the vaccine of poly (I:C) compatibility are added in this vaccine
Al., 2013,2014), in antigen submission, the function and effect of CpG are more preferable；This vaccine uses ISA201VG as oil adjuvant, with
The vaccine of ISA206 emulsification is compared to (Ren et al., 2011), other than stimulating humoral immunity, moreover it is possible to cellular immunity be stimulated to answer
It answers, extends the immune duration of protein immunization, there is availability.
Schweineseuche is a kind of deadly infectious disease as caused by swine foot-and-mouth disease virus, and infectiousness is strong, and disease incidence is high, to pig raising
Industry brings huge harm.The O-shaped aftosa prevention of China pig at present is mainly traditional inactivated vaccine and novel conjunction with vaccine
At peptide vaccine.
In terms of aftosa prevention and treatment, inactivation of virus virus leakage living not exclusively or in production of vaccine laboratory is easily caused
The outburst of FMD, and genetic engineering multi-epitope protein vaccine production process is not related to infective FMDV, there is no scattered poison
Danger can be used as a kind of new generation vaccine.
Existing to be successfully applied to field of biological pharmacy about 1. recombination E.coli high density fermentations, which has week
The advantages that phase is short, toxigenic capacity is cheap, metabolic regulation is more mature is particularly well suited for enterprise's large-scale production.
The Chinese patent of existing Publication No. CN103007273A discloses a kind of foot-and-mouth disease gene engineering mixture table
Position vaccine and preparation method thereof, the BL21-FMDV-B4 bacterial strain constructed by Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science, same
The protective epitope that multiple pedigrees are introduced in kind neoepitope Western, expands the spectrotype of neoepitope Western, to make neoepitope Western
The immune response of the induced generation of vaccine has more broad spectrum activity；By with general t cell epitope Combined design, formed composite table
Position albumen, stimulation generates more fully humoral immunity and cellullar immunologic response, and can pass through recombination E.coli high density hair
Ferment has been successfully applied to field of biological pharmacy, which has the period is short, toxigenic capacity is cheap, metabolic regulation is more mature etc.
Advantage is particularly well suited for enterprise's large-scale production.
The technology is the method for laboratory preparation, and high production cost, specific yield are small, are not suitable for industrialized production.
Our patent is the method for large-scale production, and traditional centrifugal process is changed to ultrafiltration, answers traditional dialysis
Property method be changed to dilution refolding method, overcome the carrying capacity restricted problem of large-scale instrument, equipment, substantially save production cost, manpower
It expends, produce Schweineseuche O-shaped multi-epitope protein vaccine identical with lab-quality under the premise of the production cycle, be real
Long-term aftosa prevention and control strategic objective provides a strong guarantee in existing country.
Summary of the invention
To solve problems of the prior art, the present invention provides a kind of Schweineseuche O-shaped genetic engineering multi-epitope
The production method of albumen, according to sequencing the following steps are included:
Step (1): it recombinant and inducing expression: takes in engineering bacteria seed liquor inoculation LB culture solution and ferments, send out
IPTG is added after ferment is complete and carries out inducing expression, the first recombinant protein fermentation liquor after obtaining inducing expression；
Step (2): it microorganism collection and broken: is fermented using hollow fiber column to the first above-mentioned recombinant protein being collected into
Liquid carries out concentration, then carries out break process again and obtains the second recombinant protein fermentation liquor；
Step (3): inclusion body washing is dissolved with denaturation: being sent out using buffer the second recombinant protein in hollow fiber column
Zymotic fluid is washed, then concentrated and centrifugal treating, abandons supernatant, and IB Solubilization is added after draining away the water in sediment
Buffer is mixed, and incubation at room temperature until albumen is completely dissolved, is centrifuged, the inclusion body that supernatant as slightly mentions again；
Step (4): protein purification: make above-mentioned to forgive intracorporal destination protein and occurred with filler using protein purification system
Full specific binding is cleaned unbonded foreign protein with Washing Buffer, then is washed with two kinds of different elution buffer collections
De- liquid, as destination protein, then the eluent of collection is discarded into insoluble material by centrifugation, supernatant is purifying protein
Liquid.
Step (5): purifying protein renaturation: using dilution refolding method, above-mentioned purifying protein liquid be added in renaturation solution, quiet
It sets overnight, then insoluble material is discarded by centrifugation, be concentrated, as antigen, dispensed after degerming spare using ultrafiltration membrane packet.
Preferably, in step (1), fermentation condition are as follows: 37 DEG C, PH 7.2, dissolved oxygen 40%, increasing bacterium 5 hours induce table
Up to 5 hours.
In any of the above-described scheme preferably, in step (2), the specification of the hollow fiber column is 750KD.
In any of the above-described scheme preferably, in step (3), the buffer is 1 × IB Wash of ice bath
Buffer, the IB Solubilization Buffer are 1 × IB of the N- sarcosyl containing 0.3%
SolubilizationBuffer。
In any of the above-described scheme preferably, in step (3), the condition that is centrifuged for the first time is 4 DEG C, 10000r/min from
Heart 10min, the condition of second of centrifugation are that 12000r/min is centrifuged 10min.
In any of the above-described scheme preferably, in step (4), the filler be 50% Ni-NTAHis
BindResin。
In any of the above-described scheme preferably, in step (4), NaH in the WashingBuffer2PO4Concentration is
The concentration of 100mmol/L, Tris-HCl are 10mmol/L, and the concentration of urea is 8mol/L, the pH of the WashingBuffer
It is 6.3.
In any of the above-described scheme preferably, in step (4), NaH in described two different elution buffers2PO4
Concentration is 100mmol/L, and the concentration of Tris-HCl is 10mmol/L, and the concentration of urea is 8mol/L, and pH is respectively 5.9 Hes
4.5。
In any of the above-described scheme preferably, in step (4), the condition of centrifugation is 4 DEG C, 12000r/min is centrifuged
10min。
In any of the above-described scheme preferably, in step (5), the concentration of Tris-HCl is in the renaturation solution
The concentration of 20mmol/L, DTT are 0.1mmol/L, pH 8.5.
The invention has the benefit that in terms of product quality: up to 90% or more, endotoxin is can be controlled in purity
31.25EU/mL is hereinafter, indirect hemagglutination inhibits potency in 1:210More than, on production cost and efficiency: personnel used are less, consume
Amount of reagent is less, the production cycle is shorter.
Detailed description of the invention
Fig. 1 is the SDS-PAGE result figure of albumen produced by the invention；
Fig. 2 is the Activity determination figure of albumen produced by the invention.
Specific embodiment
In order to be further understood that summary of the invention of the invention, the present invention is elaborated below in conjunction with specific embodiment.
Engineering bacteria seed liquor: BL21-FMDV-B4 is constructed and is saved by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences, egg
White peptone Sigma, yeast powder Sigma, sodium chloride traditional Chinese medicines, kanamycins solarbio, IPTG solarbio, N- dodecyl flesh
Propylhomoserin sodium solarbio, CAPS solarbio, urea traditional Chinese medicines, nickel affinity media Bo Gelong, NaH2PO4Traditional Chinese medicines, Tris-HCl
Sigma, DTT U.S. logical sequence biology, the preparation of CpG immunopotentiator Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences, ISA201VG oil assistant
Qingpu Shanghai factory of SEPPIC company of agent France T30331.
Embodiment one
Recombinant and inducing expression:
Take the engineering bacteria seed liquor 500mL being incubated overnight to be inoculated in the LB culture solution of 50L (containing 75 μ g/mL kanamycins)
In, it ferments under the conditions of 37 DEG C, PH7.2, dissolved oxygen 40%, and pass through the feed supplement of varying index fed-batch mode；IPTG is added after fermentation 5h
To final concentration of 1.0mmol/L, inducing expression 5h, the recombinant protein after obtaining inducing expression.
Microorganism collection and broken:
Concentration is carried out to the fermentation liquid being collected into using the hollow fiber column of 750KD, until stopping when 5L.Use high pressure
Concentrate is carried out break process under conditions of 900bar, two circulations by refiner.
Inclusion body washing is dissolved with denaturation:
Broken fermentation liquid is washed using the hollow fiber column of 750KD, buffer is 1 × IB of ice bath
WashBuffer(EDTA 10mmol/L；Tris-HCl 20mmol/L,pH7.5；TritonX-1001%), 5 are replaced in equal volume
Times, half volume is concentrated after being replaced.4 DEG C, centrifugation 10min collects inclusion body under the conditions of 10000g, abandon supernatant, drain the water
It weighs after point, and 1 × IB of the N- sarcosyl containing 0.3% is added by 1:100
SolubilizationBuffer (50mmol/L CAPS, pH 11.0), mixes gently, blows and beats repeatedly, is incubated at room temperature 30min,
Time to albumen can be appropriately extended to be completely dissolved, 12000r/min centrifugation 10min discards insoluble material, and supernatant as slightly mentions
Inclusion body.
Protein purification:
Using protein purification system, by filler (50% Ni-NTAHisBindResin) and inclusion body protein lysate
Volume ratio be that 4:1 fills column, and makes destination protein and Ni-NTAHisBindResin that complete specific binding occur, use
Washing Buffer(100mmol/L NaH2PO4, 10mmol/L Tris-HCl, 8mol/L urea；PH6.3 it) cleans and does not tie
Foreign protein is closed, then with different pH value (5.9 and 4.5) elution buffer (100mmol/LNaH2PO4, 10mmol/L Tris-HCl,
8mol/L urea) collect eluent, as destination protein.4 DEG C, 12000r/min centrifugation 10min discard insoluble material, on
It is clearly purifying protein liquid.
Purifying protein renaturation:
Using dilution refolding method, purifying protein liquid is slowly added into 3 times of its renaturation solution (20mmol/L dropwise
Tris-HCl, pH8.5 DTT containing 0.1mmol/L) in, it stands overnight.4 DEG C, 12000r/min centrifugation 10min discard it is insoluble
Substance.Concentrating it to protein concentration using 10KD ultrafiltration membrane packet is 1.0mg/mL, as antigen, is dispensed after degerming spare.
Embodiment two
Recombinant and inducing expression:
Take the engineering bacteria seed liquor 400mL being incubated overnight to be inoculated in the LB culture solution of 50L (containing 75 μ g/mL kanamycins)
In, it ferments under the conditions of 37 DEG C, PH7.2, dissolved oxygen 40%, and pass through the feed supplement of varying index fed-batch mode；IPTG is added after fermentation 5h
To final concentration of 1.0mmol/L, inducing expression 5h, the recombinant protein after obtaining inducing expression.
Microorganism collection and broken:
Concentration is carried out to the fermentation liquid being collected into using the hollow fiber column of 750KD, until stopping when 5L.Use high pressure
Concentrate is carried out break process under conditions of 900bar, two circulations by refiner.
Inclusion body washing is dissolved with denaturation:
Broken fermentation liquid is washed using the hollow fiber column of 750KD, buffer is 1 × IB of ice bath
WashBuffer(EDTA 10mmol/L；Tris-HCl 20mmol/L,pH7.5；TritonX-1001%), 5 are replaced in equal volume
Times, half volume is concentrated after being replaced.4 DEG C, centrifugation 10min collects inclusion body under the conditions of 10000g, abandon supernatant, drain the water
Point after weigh, and by 1:100 be added containing 0.3% N- sarcosyl 1 ×
IBSolubilizationBuffer (50mmol/L CAPS, pH 11.0), mixes gently, blows and beats repeatedly, incubation at room temperature
30min can be appropriately extended time to albumen and be completely dissolved, and 12000r/min centrifugation 10min discards insoluble material, and supernatant is
For the inclusion body slightly mentioned.
Protein purification:
Using protein purification system, by filler (50% Ni-NTAHisBindResin) and inclusion body protein lysate
Volume ratio be that 4:1 fills column, and makes destination protein and Ni-NTAHisBindResin that complete specific binding occur, use
WashingBuffer(100mmol/L NaH2PO4, 10mmol/L Tris-HCl, 8mol/L urea；PH6.3 it) cleans unbonded
Foreign protein, then with different pH value (5.9 and 4.5) elution buffer (100mmol/LNaH2PO4, 10mmol/L Tris-HCl,
8mol/L urea) collect eluent, as destination protein.4 DEG C, 12000r/min centrifugation 10min discard insoluble material, on
It is clearly purifying protein liquid.
Purifying protein renaturation:
Using dilution refolding method, purifying protein liquid is slowly added into 3 times of its renaturation solution (20mmol/L dropwise
Tris-HCl, pH8.5 DTT containing 0.1mmol/L) in, it stands overnight.4 DEG C, 12000r/min centrifugation 10min discard it is insoluble
Substance.Concentrating it to protein concentration using 10KD ultrafiltration membrane packet is 1.0mg/mL, as antigen, is dispensed after degerming spare.
Embodiment three
Recombinant and inducing expression:
Take the engineering bacteria seed liquor 300mL being incubated overnight to be inoculated in the LB culture solution of 50L (containing 75 μ g/mL kanamycins)
In, it ferments under the conditions of 37 DEG C, PH7.2, dissolved oxygen 40%, and pass through the feed supplement of varying index fed-batch mode；IPTG is added after fermentation 5h
To final concentration of 1.0mmol/L, inducing expression 5h, the recombinant protein after obtaining inducing expression.
Microorganism collection and broken:
Concentration is carried out to the fermentation liquid being collected into using the hollow fiber column of 750KD, until stopping when 5L.Use high pressure
Concentrate is carried out break process under conditions of 900bar, two circulations by refiner.
Inclusion body washing is dissolved with denaturation:
Broken fermentation liquid is washed using the hollow fiber column of 750KD, buffer is 1 × IB of ice bath
WashBuffer(EDTA 10mmol/L；Tris-HCl 20mmol/L,pH7.5；TritonX-1001%), 5 are replaced in equal volume
Times, half volume is concentrated after being replaced.4 DEG C, centrifugation 10min collects inclusion body under the conditions of 10000g, abandon supernatant, drain the water
It weighs after point, and 1 × IB of the N- sarcosyl containing 0.3% is added by 1:100
SolubilizationBuffer (50mmol/L CAPS, pH 11.0), mixes gently, blows and beats repeatedly, is incubated at room temperature 30min,
Time to albumen can be appropriately extended to be completely dissolved, 12000r/min centrifugation 10min discards insoluble material, and supernatant as slightly mentions
Inclusion body.
Protein purification:
Using protein purification system, by filler (50% Ni-NTAHisBindResin) and inclusion body protein lysate
Volume ratio be that 4:1 fills column, and makes destination protein and Ni-NTAHisBindResin that complete specific binding occur, use
WashingBuffer(100mmol/L NaH2PO4, 10mmol/L Tris-HCl, 8mol/L urea；PH6.3 it) cleans unbonded
Foreign protein, then with different pH value (5.9 and 4.5) elution buffer (100mmol/LNaH2PO4, 10mmol/L Tris-HCl,
8mol/L urea) collect eluent, as destination protein.4 DEG C, 12000r/min centrifugation 10min discard insoluble material, on
It is clearly purifying protein liquid.
Purifying protein renaturation:
Using dilution refolding method, purifying protein liquid is slowly added into 3 times of its renaturation solution (20mmol/L dropwise
Tris-HCl, pH8.5 DTT containing 0.1mmol/L) in, it stands overnight.4 DEG C, 12000r/min centrifugation 10min discard it is insoluble
Substance.Concentrating it to protein concentration using 10KD ultrafiltration membrane packet is 1.0mg/mL, as antigen, is dispensed after degerming spare.
Experimental result:
1.SDS-PAGE result is as shown in Figure 1.
2. protein active (reverse blood clotting inhibition) result is as shown in Fig. 2, wherein A, B are positive control (A type), C is sun
Property control (O-shaped), 1. number D behavior sample is (before crossing nickel column renaturation), E behavior sample 2. number (after not crossing nickel column renaturation), F behavior
3. number (after crossing nickel column renaturation), G is negative control to sample.
Upstream fermentation: (engineering bacteria → inclusion body)
Downstream purification: (inclusion body → antigen)
3. comparison:
(1) cell density of shaking flask culture and fermentation tank culture, activity
It uses shaking flask culture: being packed into 500mL culture medium in the shaking flask of 2L volume.It finally harvests after cultivating 12h to bacterium solution
OD600 value is 2,900,000,000/mL up to 3.318, viable bacteria number；Every 1L bacterium solution available wet bacterium weight after centrifugal treating is 4.83g,
The weight of forgiving that can finally harvest is 3.28g.
It uses fermentation tank culture: being packed into 3.5L culture medium in the shaking flask of 5L volume.It finally harvests after cultivating 12h to bacterium solution
OD600 value is 36,000,000,000/mL up to 25.79, viable bacteria number；Every 1L bacterium solution available wet bacterium weight after centrifugal treating is
58.9g, the weight of forgiving that can finally harvest is 40.17g.
(2) antigen active of the bacterium of shaking flask culture and fermentation tank culture after downstream processing
Use shaking flask culture: 1:210, use fermentation tank culture: 1:212, 1:4096 is increased to from 1:1024；Albumen is pure
Degree and endotoxin content are both qualified.
It is significantly improved in addition, the method has in quantity of sample handling and processing time.Conventional method is centrifugal process, processing
Measure that small, time-consuming, expense liquid is more and sample batch is difficult to ensure often a batch of sample stable homogeneous too much.Now mostly using super
Filter method, hollow fiber column or film packet, treating capacity is big, time-consuming is short, saves liquid and sample stable homogeneous.
It will be apparent to those skilled in the art that the production of Schweineseuche O-shaped genetic engineering multi-epitope albumen of the invention
Method method include aforementioned present invention specification summary of the invention and specific embodiment part and attached drawing shown by each portion
Any combination divided describes one by one as space is limited and for each scheme for keeping specification concise without constituting these combinations.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in guarantor of the invention
Within the scope of shield.
Claims (10)
1. a kind of production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen, according to sequencing the following steps are included:
Step (1): it recombinant and inducing expression: takes in engineering bacteria seed liquor inoculation LB culture solution and ferments, fermented
IPTG is added afterwards and carries out inducing expression, the first recombinant protein fermentation liquor after obtaining inducing expression；
Step (2): microorganism collection and broken: using hollow fiber column to the first above-mentioned recombinant protein fermentation liquor being collected into
Then row concentration carries out break process again and obtains the second recombinant protein fermentation liquor；
Step (3): inclusion body washing is dissolved with denaturation: using buffer to the second recombinant protein fermentation liquor in hollow fiber column
It is washed, then concentrated and centrifugal treating, abandons supernatant, IB Solubilization is added after draining away the water in sediment
Buffer is mixed, and incubation at room temperature until albumen is completely dissolved, is centrifuged, the inclusion body that supernatant as slightly mentions again；
Step (4): protein purification: using protein purification system make it is above-mentioned forgive intracorporal destination protein and filler occur it is complete
Specific binding cleans unbonded foreign protein with Washing Buffer, then collects eluent with two kinds of different elution buffers,
As destination protein, then the eluent of collection is discarded into insoluble material by centrifugation, supernatant is purifying protein liquid.
Step (5): purifying protein renaturation: dilution refolding method is used, above-mentioned purifying protein liquid is added in renaturation solution, was stood
Night, then insoluble material is discarded by centrifugation, it is concentrated, as antigen, is dispensed after degerming spare using ultrafiltration membrane packet.
2. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In, in step (1), fermentation condition are as follows: 37 DEG C, PH 7.2, dissolved oxygen 40%, increasing bacterium 5 hours, inducing expression 5 hours.
3. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In in step (2), the specification of the hollow fiber column is 750KD.
4. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In in step (3), the buffer is 1 × IB Wash Buffer, the IB Solubilization Buffer of ice bath
For 1 × IB Solubilization Buffer of the N- sarcosyl containing 0.3%.
5. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In in step (3), the condition being centrifuged for the first time is 4 DEG C, 10000r/min is centrifuged 10min, and the condition of second of centrifugation is
12000r/min is centrifuged 10min.
6. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In, in step (4), the filler be 50% Ni-NTAHisBindResin.
7. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In, in step (4), NaH in the Washing Buffer2PO4Concentration is 100mmol/L, and the concentration of Tris-HCl is
The concentration of 10mmol/L, urea are 8mol/L, and the pH of the WashingBuffer is 6.3.
8. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In, in step (4), NaH in described two different elution buffers2PO4Concentration is 100mmol/L, and the concentration of Tris-HCl is
The concentration of 10mmol/L, urea are 8mol/L, and pH is respectively 5.9 and 4.5.
9. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In in step (4), the condition of centrifugation is 4 DEG C, 12000r/min is centrifuged 10min.
10. the production method of Schweineseuche O-shaped genetic engineering multi-epitope albumen according to claim 1, feature exist
In in step (5), the concentration of Tris-HCl is 20mmol/L in the renaturation solution, and the concentration of DTT is 0.1mmol/L, and pH is
8.5。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113136407A (en) * | 2021-05-26 | 2021-07-20 | 武汉华美生物工程有限公司 | Renaturation method of inclusion body and kit |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602958A (en) * | 2003-09-29 | 2005-04-06 | 厦门厦大建南应用技术有限公司 | A and O type gene engineering bivalent polypeptide vaccine of livestock foot-and-mouth disease virus and its preparation process |
| CN1814757A (en) * | 2005-02-03 | 2006-08-09 | 中国农业科学院兰州兽医研究所 | Novel CpGDNA adjurar, its preparing method and vaccine containing same |
| CN103007273A (en) * | 2012-11-16 | 2013-04-03 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease genetic engineering mixed epitope vaccine and preparation method thereof |
| CN103993058A (en) * | 2014-05-29 | 2014-08-20 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Method for extracting and purifying recombinant human endostatin |
| CN104788547A (en) * | 2015-04-23 | 2015-07-22 | 吕宏亮 | Foot-and-mouth disease virus 2C3ABC recombinant protein as well as preparation method and application thereof |
| CN105968182A (en) * | 2016-06-03 | 2016-09-28 | 黄文林 | Production process of recombinant human cryptochrome protein I (hCRY1) and composition thereof |
-
2018
- 2018-10-08 CN CN201811166475.2A patent/CN109266710A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602958A (en) * | 2003-09-29 | 2005-04-06 | 厦门厦大建南应用技术有限公司 | A and O type gene engineering bivalent polypeptide vaccine of livestock foot-and-mouth disease virus and its preparation process |
| CN1814757A (en) * | 2005-02-03 | 2006-08-09 | 中国农业科学院兰州兽医研究所 | Novel CpGDNA adjurar, its preparing method and vaccine containing same |
| CN103007273A (en) * | 2012-11-16 | 2013-04-03 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease genetic engineering mixed epitope vaccine and preparation method thereof |
| CN103993058A (en) * | 2014-05-29 | 2014-08-20 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Method for extracting and purifying recombinant human endostatin |
| CN104788547A (en) * | 2015-04-23 | 2015-07-22 | 吕宏亮 | Foot-and-mouth disease virus 2C3ABC recombinant protein as well as preparation method and application thereof |
| CN105968182A (en) * | 2016-06-03 | 2016-09-28 | 黄文林 | Production process of recombinant human cryptochrome protein I (hCRY1) and composition thereof |
Non-Patent Citations (2)
| Title |
|---|
| YIMEI CAO ET AL: "Poly(I:C) combined with multi-epitope protein vaccine completely protects against virulent foot-and-mouth disease virus challenge in pigs", 《ANTIVIRAL RESEARCH》 * |
| 刘斌等: "简述包涵体表达口蹄疫基因工程疫苗下游工艺", 《甘肃畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113136407A (en) * | 2021-05-26 | 2021-07-20 | 武汉华美生物工程有限公司 | Renaturation method of inclusion body and kit |
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